INSULIN STIMULATION OF THE FATTY-ACID SYNTHASE PROMOTER IS MEDIATED BY THE PHOSPHATIDYLINOSITOL 3-KINASE PATHWAY - INVOLVEMENT OF PROTEIN-KINASE PATHWAY

Fatty acid synthase (FAS) is a critical enzyme in de novo lipogenesis. It catalyzes the seven steps in the conversion of malonyl-CoA and ace tyl-CoA to palmitate, We have shown that the rate of FAS transcription is induced dramatically when fasted animals are refed with a high car bohydrate, fat-free diet or when streptozotocin-diabetic mice are give n insulin. The FAS promoter was up-regulated by insulin through the pr oximal insulin response sequence containing an E-box motif at the -65- base pair position. Binding of upstream stimulatory factors to the -65 E-box is functionally required for insulin regulation of the FAS prom oter. In the present study, we characterized signaling pathways in the insulin stimulation of FAS transcription using specific inhibitors fo r various signaling molecules and transfecting engineered phosphatidyl inositol (PI) 3-kinase subunits and protein kinase B (PKB)/Akt, PD9805 9 and rapamycin, which inhibit MAP kinase and P70 S6 kinase, respectiv ely, had little effect on the insulin-stimulated FAS promoter activity in 3T3-L1 adipocytes, On the other hand, wortmannin and LY294002, whi ch specifically inactivate PI 3-kinase, strongly inhibited the insulin -stimulated FAS promoter activity. As shown in RNase protection assays , LY294002 also inhibited insulin stimulation of the endogenous FAS mR NA levels in 3T3-L1 adipocytes, Cotransfection of expression vectors f or the constitutively active P110 subunit of PI 3-kinase resulted in a n elevated FAS promoter activity in the absence of insulin and a loss of further insulin stimulation. Transfecting a dominant negative P85 s ubunit of PI 3-kinase decreased FAS promoter activity and blocked insu lin stimulation. Furthermore, cotransfected wild-type PKB/Akt increase d FAS promoter activity in the absence of insulin and a loss of insuli n responsiveness of the FAS promoter. On the other hand, kinase-dead P KB/Akt acted in a dominant negative manner to decrease the FAS promote r activity and abolished its insulin responsiveness. These results dem onstrate that insulin stimulation of fatty acid synthase promoter is m ediated by the PI 3-kinase pathway and that PKB/Abt is involved as a d ownstream effector.